The ultimate expression of the genetic makeup of a cell resides in the proteins of that cell. Although protein synthesis is a complex process requiring numerous proteins and nucleicacids, to a large extent the specificity is determined by the first step of the process - the activation and transfer of an amino acid to its respective transfer RNA. Although considerable information about aminoacyl-tRNA synthetases and tRNA has been obtained, relatively little is known about the control of protein synthesis that may be exercised at the level of aminoacyl-tRNA formation. Our long term goal is the elucidation of the pathways, enzymes and regulatory factors which determine the levels of tRNA and synthetases in cells, and their effect on the various cellular processes in which aminoacyl-tRNA participates. The studies we propose should provide needed information in this central area of macromolecular metabolism. Our specific aims are: 1) to identify, purify and determine the physiological role of nucleases involved in tRNA processing and end-turnover of tRNA; 2) to clone, sequence and obtain high level expression of the cca gene of E. coli in order to examine protein-nucleic acid interactions in the tRNA-tRNA nucleotidyltransferase system; 3) to study the structural organization of mammalian aminoacyl-tRNA synthetases with particular emphasis on the role of lipids and membranes and protein modification for formation of the high molecular weight synthetase complex; and 4) to determine how tRNA levels are regulated with regard to codon usage and to maintenance of stoichiometry with synthetases. The methods to be used will be enzymological, structural and genetic. In view of the central role of proteins in all cell functions, and the importance of aminoacyl-tRNA formation for the specificity of this process, an understanding of the mechanism and regulation of this step is of great importance. Elucidation of these aspects of protein synthesis in normal cells is a prerequisite to any understanding of abnormal situations which may occur under pathological conditions.